As retinal axons grow along the retinotectal pathway from their origins in the retina to their final targets in the tectum, they are guided by a complex array of signals in the extracellular matrix, and by interactions with other retinal axons. This process is not only critical for the development of visual function, but serves as an accessible model for axon guidance in general. This proposal will use genetic, embryological, and imaging approaches in the zebrafish embryo to analyze retinal axon guidance in vivo, studying how Slit- Robo signaling and axon-axon interactions act to shape the retinotectal projection. We will address three main questions about Slit-Robo signaling. First, can Slits change from repulsive to attractive in different contexts as growth cones pass from one part of their pathway to another? Expression studies, time lapse analysis, in vitro assays, and a novel in vivo method for targeted misexpression will address this question definitively. Second, do different cytoplasmic domains of the Robo2 receptor have different required functions? And third, is the alternative splicing that we have seen in the robo2 gene functionally significant? We will address these two questions by employing splice-blocking morpholino oligonucleotides for a powerful new strategy, targeted exon deletion. Within the retina, our preliminary data show that early-born central RGCs are required for later-born peripheral axons to exit the eye. We will first comprehensively analyze retinal axon pathfinding within the retina, then use time lapse and transplant experiments to ask how early RGCs act to guide later axons. Finally, we will test which cell-adhesion molecules mediate retinal axon fasciculation, and what role they play in guiding retinal axons within the retina, in the chiasm, and in the optic tract. Relevance to public health. Visual system anatomy and genetic control mechanisms are both highly conserved across the vertebrates from fish to humans. Understanding the molecular mechanisms that work together to assemble the zebrafish visual system is thus directly relevant to human development. Furthermore, our data showing the importance of axon-axon interactions during development highlight the necessity of considering such interactions during axon regeneration in response to injury or disease.